The angiogenic switch plays a fundamental role in tumor vascularization and metastasis;however, the underlying cellular and molecular mechanisms by which microenvironmental conditions regulate this process are still unclear. This project will address the hypothesis that the creation of distinct tumor-like niches and crosstalk between cells residing within these niches up-regulates expression of pivotal tumor cytokines that contribute to the recruitment of bone marrow-derived endothelial progenitor cells (EPCs) and enhanced tumor angiogenesis. To address our hypothesis we will utilize a physical-sciences based cancer biology approach that combines 3-D cell culture, microfluidics, and mathematical modeling. Our study design is based on 4 aims: In aim 1, we will design 3-D microfluidic tumor cultures that will allow us to test the hypothesis that the signaling of soluble factors, as regulated by spatially resolved differences in oxygen tension, provides a paracrine mechanism that spans between niches to regulate the differential expression of cytokines by both tumor and stromal cells. In aim 2, we will evaluate whether the global and local dynamics of tumor and stromal cell signaling, as elucidated in aim 1, impact invasion angiogenesis. To this end, we will expand the microfluidic platform developed in aim 1 to integrate endothelialized microchannels. This system will be remodelable and can be adjusted to exhibit enhanced matrix stiffness as typical of the tumor stroma. In aim 3, we will determine whether physicochemically mediated changes in neovessel formation as defined in aim 2 lead to the formation of vascular niches that impact the phenotypic identity, spatial and temporal contribution, and biological function of EPCs and their role in tumor angiogenesis. Finally, in aim 4, we will conduct dynamic global transcriptome and epigenome analysis of EPCs that have incorporated in the microfluidic microvessels. The generated data will be incorporated into computational signal transduction network analysis to identify molecular targets that may be responsible for physicochemically mediated changes in the angiogenic switch. Our proposed studies have the potential to improve current strategies of anti-angiogenic therapies by enhancing our understanding of the tumor angiogenic switch and identifying molecular mechanisms that may be involved in this process and provide therapeutically relevant targets. PUBLIC HEALTH RELEVANCE: Tumor angiogenesis represents a critical event of cancer that involves the recruitment of bone marrow derived endothelial progenitor cells;however, the exact mechanisms and effects by which microenvironmental conditions regulate these processes are not well understood. Using micropathological 3-D tumor models and mathematical modeling approaches this research will address the hypothesis that the creation of oxygen dependent tumor niches, and paracrine cellular crosstalk between these niches, up-regulates expression of pivotal tumor cytokines that impact invasion angiogenesis and the recruitment of endothelial progenitor cells. This interdisciplinary strategy has the potential to revolutionize our understanding of tumor vascularization and elucidate fundamentally new mechanisms of pro-angiogenic activity in cancers that could form a basis for improved anti-angiogenic therapies and clinical outcomes.